Background: Tumor mutational burden (TMB) may be a predictive marker for response to anti-PD1/PDL1 agents (IO). Molecular heterogeneity of various biomarkers for GEA has been established. To characterize heterogeneity of TMB and its clinical relevance, we compared TMB in primary (1⁰) & metastatic (met) tumors at baseline newly diagnosed stage IV advanced GEA (aGEA), and before & after chemotherapy treatment (tx) for stage II–IV GEA. We assessed the prognostic relevance of TMB in aGEA. Methods: We retrospectively reviewed a cohort of 127 patients (pts) diagnosed with GEA in 2012–2019, for a total of 280 tumor samples with TMB data. TMB level was defined as low (≤5/Mb), intermediate (int) ( > 5/Mb, ≤15/Mb), or high (hi) (≥15/Mb), determined by Foundation One. Analysis was performed by Fisher’s exact test for PDL1/TMB, McNemar’s test for paired TMB, and univariate Cox proportional-hazards model for overall survival (OS). Results: Of 280 tumors, 50% (140/280) had low TMB, 45% (125/280) int TMB, & 5% (15/280) hi TMB. TMB ranged 0–58.6/Mb (median 5.3/Mb). Of tumors with hi TMB, 53% (8/15) were MSI-Hi, while of MSI-Hi tumors, 100% (8/8) were TMB hi. TMB level did not correlate with PDL1 status (p= 0.83). Concordance between TMB levels of paired baseline 1⁰ and baseline met tumors was 66% (29/44) (Table). TMB level was lower in the met than in the 1⁰ in 23% (10/44) of cases, and higher in the met in 11% (5/44). Of 4 TMB hi baseline 1⁰ tumors, 2 were not TMB hi in the met; of 40 TMB low/int baseline 1⁰ tumors, 0 were TMB hi in the met (p= 0.16). Post-tx tumors exhibited 71% (42/59) concordance of TMB levels compared to pre-tx 1⁰ tumors. Of 2 TMB hi baseline tumors, 1 was not TMB hi in the post-tx tumor; of 57 TMB low/int baseline tumors, 0 were TMB hi in the post-tx tumor (p= 0.32). In pts with aGEA at diagnosis, OS did not significantly differ depending on baseline 1⁰ tumor TMB level (median OS of 21.4 [95% CI 15.4–27.9] months for TMB low, 14.6 [10.9–23.5] months for TMB int, and 9.6 [3.9–NA] for TMB hi; p= 0.3), nor depending on baseline met TMB level. Conclusions: Notable baseline spatial discordance of TMB was observed, particularly TMB hi 1⁰ to low/int met. Discordance was also observed before & after tx, without significant increase towards TMB hi temporally. Spatiotemporal heterogeneity may impact the role of TMB as a predictive biomarker & warrants further study.